Hexyl isobutyrate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 2018 to June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hexyl isobutyrate

EC Number:

219-075-8

EC Name:

Hexyl isobutyrate

Cas Number:

2349-07-7

Molecular formula:

C10H20O2

IUPAC Name:

hexyl 2-methylpropanoate

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

*Specification:
- EpiDermTM-Kit, procured by MatTek In Vitro Life Science Laboratories, Bratislava (EPI-200-SIT)
- EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis.
- EpiDermTM tissues are cultured on specially prepared cell culture inserts.

*Pre - Tests:
1. Nylon mesh compatibility
- 30 µL of the test item were pipetted onto a nylon mesh on a microscope slide.
- Reaction with the nylon mesh after an exposure time of 1 hour is monitored

2. Assessment of Coloured or Staining Test Items
- Test if test item develops a colour without MTT addition.
- 30 µL test item were given in a test tube with 0.3 mL H2O demin. and incubated at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour.

3. Assessment of Direct Reduction of MTT by the Test Item
- 30 µL test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour.
- Untreated MTT medium was used as control.
- if direct MTT reduction by the test item had not taken place and no data correction is necessary.

4. Pre-Incubation of Tissues
- For each treatment group (negative control, test item and positive control) a 6-well-plate was prepared with 0.9 mL assay medium in 3 of the 6 wells (upper row).
- Tissues were inspected for viability.
- Tissues were transferred into the wells, which contain medium by using sterile forceps and placed into the incubator at 37 ± 1°C and 5 ± 0.5% CO2 for 1 hour.
- After 1 hour pre-incubation, the other 3 wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every viable tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 ± 1°C and 5.0 ± 0.5% CO2 for 18 hours and 55 minutes.

*Treatment:
- One plate (3 tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
- One plate was used as positive control; each tissue was treated with 30 µL 5% SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
- One plate was used for treatment with the test item:
30 µL test item were applied, and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
- Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.
- 1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
- After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).
Then, the tissues were set in the incubator for 23 hours and 25 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.

*Medium Renewal:
- After post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm).
- 0.9 mL assay medium were filled in the lower row of the 6-well-plate.
- The inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 19 hours and 15 minutes for post-incubation at 37 ± 1°C and 5.0 ± 0.5% CO2.

*MTT Assay:
- After a total incubation time of 42 hours and 40 minutes, a 24-well-plate was prepared with 300 µL freshly prepared MTT-solution in each well.
- The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 hours at 37 ± 1°C and 5.0 ± 0.5% CO2.
- After this time, the MTT-solution was aspirated and replaced by DPBS buffer. This was then aspirated, too, and replaced several times.
- At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate.
- Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken (120 rpm) for 2 hours at room temperature.
- After 2 hours, the inserts were pierced with an injection needle, taking care that all colour was extracted.
- The inserts were then discarded and the content of each well was thor-oughly mixed in order to achieve homogenisation.
- From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate which was read in a plate spectrophotometer at 570 nm.

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): undiluted


NEGATIVE CONTROL
- Dulbecco’s Phosphate Buffered Saline (DPBS buffer without CaCl2 and without MgCl2)
- Amount(s) applied (volume or weight): 30 µL


POSITIVE CONTROL
- Sodium dodecyl sulphate (SDS), CAS No. 151-21-3, solution in demineralised water containing 5% SDS
- Amount(s) applied (volume or weight): 30 µL

Duration of treatment / exposure:

60 min at 37 ± 1°C and 5.0 ± 0.5% CO2.

Duration of post-treatment incubation (if applicable):

in the incubator for 23 hours and 25 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

102.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The skin irritation potential of hexyl isobutyrate was determined in reconstructed human epidermis ( EpiDermTM-Kit, procured by MatTek In Vitro Life Science Laboratories, Bratislava (EPI-200-SIT)).
After the treatment with the pure test item hexyl isobutyrate, the mean value of relative tissue viability was increased to 102.9%. This value is above the threshold for skin irritation potential (50% noTest items that induce values above the threshold of 50% are considered non-irritant to skin.
Theredore , hexyl isobutyrate is considered non-orritant to skin in the Reconstructed human Epidermis (Rhe) Test Method.

Executive summary:

The Skin Irritation Potential of hexyl isobutyrate was determined using the Reconstructed human Epidermis (RhE) Test Method following EU-Method B.46 and OECD 439.

One valid experiment was performed.

Tissue of the human skin model EpiDerm^TMwas treated with hexyl isobutyrate for 60 minutes. Three replicates were used for chemical treatment and for the controls.

The test item was applied directly to each tissue and spread to uniformly cover the tissue surface (0.63 cm²).

DPBS-buffer was used as negative control and 5% SDS solution was used as positive control.

After treatment with the negative control, the mean absorbance values was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.3. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 2.5% (required: ≤ 20%)

The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%). For these reasons, the result of the test is considered valid.

 

After the treatment with the test item, the mean value of relative tissue viability was increased to 102.9%. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin.

 

Therefore, hexyl isobutyrate is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.